Biochemistry 1995, 34, 13642- 13650
13642
Oxidation of Kinetically Trapped Thiols by Protein Disulfide Isomerase? Kenneth W. Walker and Hiram F. Gilbert* The Verna and Marrs McLean Department of Biochemistry, Baylor College of Medicine, Houston, Texas 77030 Received June 5, 1995; Revised Manuscript Received August 1, 1995@
ABSTRACT: The formation of a stabilized structure during oxidative protein folding can severely retard disulfide formation if the structure must be disrupted to gain access to buried cysteines. These kinetic traps can slow protein folding and disulfide bond formation to the extent that unassisted folding is too slow to be kinetically competent in the cell. Protein disulfide isomerase (PDI) facilitates the oxidation of a kinetically trapped state of RTEM-1 p-lactamase in which two cysteines that form the single disulfide bond in the native protein are buried and approximately 500-fold less reactive than exposed cysteines. Under second-order conditions, PDI-dependent oxidation of reduced, folded P-lactamase is 500-fold faster than GSSG-dependent oxidation. The rate difference observed between PDI and GSSG can be accounted for by the 520-fold higher kinetic reactivity of PDI as an oxidant. Noncovalent interactions between PDI (35 pM) and P-lactamase increase the reactivity or unfolding of P-lactamase in the steady-state by less than 3-fold. At high concentrations of PDI or alkylating agents, the reaction of P-lactamase cysteines approaches a constant rate, limited by the spontaneous unfolding of the protein (kunfold = 0.024 f 0.005 min-l). PDI does not substantially increase the rate of P-lactamase unfolding; however, once P-lactamase spontaneously unfolds, PDI at concentrations greater than 44 f 4 pM, oxidizes the unfolded substrate before it can refold (kfold = 1.5 =t0.2 min-l). PDI also facilitates the glutathione disulfide-dependent oxidation of P-lactamase by approximately 1.7-3-fold, even at saturating GSSG concentrations; however, oxidation rates never exceed the rate of spontaneous P-lactamase unfolding. The high kinetic reactivity of PDI as an oxidant and the almost millimolar concentrations of PDI found in the endoplasmic reticulum provide one mechanism to avoid kinetic traps by making oxidation kinetically competitive with the formation of kinetic traps. In addition, PDI's high reactivity and its noncovalent interactions with unfolded proteins may minimize the significance of even relatively stable kinetic traps, as long as unfolding of the kinetic trap does not become rate-limiting. Chaperones, including the hsp70 chaperone of the endoplasmic reticulum, BiP, and the Escherichia coli chaperonin, GroEL, do not appear to increase the rate of P-lactamase unfolding. This suggests that the unfolding rate may help define those structures that the endoplasmic reticulum quality control system recognizes as native.
In the absence of folding catalysts and chaperones, the folding of disulfide-containing proteins is often slow (Goldberger et al., 1963). Even under optimal conditions, the uncatalyzed oxidative refolding of reduced ribonuclease (RNase)' has a half-life of about 1.5 h (Lyles & Gilbert, 1990a), and bovine pancreatic trypsin inhibitor (BPTI) refolds even more slowly (t1/2 = 8 h) (Weissman & Kim, 1993). In the endoplasmic reticulum, where disulfide-containingproteins are folded in eukaryotic cells, the time available for folding and exit from the ER is typically about 0.5-1.5 h (Lodish et al., 1983). Clearly, spontaneous oxidative folding is often not fast enough to be kinetically competent in vivo. It was this realization that inspired the first isolation of a cellular catalyst of oxidative folding, protein disulfide isomerase (PDI) (Goldberger et al., 1963). During protein folding, cysteines that must eventually be oxidized to form disulfides in the native structure may +
Supported by NIH Grant GM40379.
* Td whom cbrrespondence should be addressed [telephone (7 13)
798-5880, FAX (713) 796-94381. Abstract published in Advan& ACS Abstracts, September 15, 1995. i Abbreviations: PDI, protein disulfide isomerase; GSH, glutathione; GSSG, glutathione disulfide; PLa, RTEM- 1 P-lactamase; PLa,d, reduced and refolded P-lactamase; ER, endoplasmic reticulum; RNase, bovine pancreatic ribonuclease A; BPTI, bovine pancreatic trypsin inhibitor; Gdn-HC1, guanidine hydrochloride; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis. @
become trapped in the reduced state by the formation of stabilized native or non-native structures, including those stabilized by other disulfide bonds in the molecule (Goto & Hamaguchi, 1981; States et al., 1984; Creighton & Goldenberg, 1984; Weissman & Kim, 1993). In order to complete folding, these kinetically trapped intermediates must unfold sufficiently to expose sterically shielded cysteine residues to oxidants (including intramolecular oxidants) or they must rearrange to species with accessible cysteines. Weissman and Kim (1993) reported that PDI accelerates the rearrangement of kinetically trapped BPTI folding intermediates with buried sulfhydryls by 4000to 6000-fold compared to the approximately 140-fold rate enhancement observed in PDI-catalyzed folding of RNase (Lyles & Gilbert, 1990b). However, for both BPTI and RNase, the kcat'sof the PDI-dependent reaction are similar, 0.3-5 min-' (Weissman & Kim, 1993). Thus, the differences in the rate acceleration for these two proteins are largely due to differences in the spontaneous oxidative folding rates rather than the rate of the PDI-catalyzed reaction. The comparable rates of folding observed for the PDI-catalyzed reactions suggest that PDI might provide some mechanism for destabilizing the kinetic traps encountered during spontaneous refolding and minimizing their influence on slow refolding.
0006-2960/95/0434-13642$09.00/0 0 1995 American Chemical Society
Oxidation of Kinetically Trapped Thiols PDI has several properties that might be useful in resolving potential problems with kinetic traps. PDI is a 55 kDa, ER resident that catalyzes disulfide bond formation and rearrangement (Freedman, 1989; Noiva & Lennarz, 1992). It is an abundant protein; concentrations in the ER have been estimated at near millimolar amounts (Lyles & Gilbert, 1990b; Zapun et al., 1992). The two active sites of the molecule, including the sequence WCGHCK, are located in two thioredoxin-homology domains found near the N- and C-termini (Edman et al., 1985). The disulfide bonds of PDI are exceptionally good oxidizing agents with a high redox potential (Lyles & Gilbert, 1990a; Hawkins & Freedman, 1991; Lundstrom & Holmgren, 1993) and a high kinetic reactivity with thiols (Gilbert, 1989; Darby et al., 1994). In addition to the redox-active dithioVdisulfide centers at the active sites (Lyles & Gilbert, 1994; Vuori et al., 1992), PDI interacts with unfolded proteins and peptides at a relatively weak binding site that exhibits little sequence preference (Morjana & Gilbert, 1991; Noiva et al., 1993). PDI is an essential protein in yeast, but a mutant PDI with undetectable disulfide isomerase activity can rescue the null mutation, suggesting that some activity other than disulfide formation and rearrangement is essential for yeast viability (LaMantia & Lennarz, 1993). In order to explore mechanisms by which PDI might accelerate the oxidation of sterically inaccessible cysteines, we have exploited a simple model system, RTEM-1 ,&lactamase @La). Native pLa is a monomer with only one disulfide bond, so reduction or isomerization of other disulfides in the molecule does not complicate the mechanism. The single disulfide contributes minimally to stability, and even under reducing conditions, pLa spontaneously refolds to a stable conformation @Lared) with near-native enzymatic activity (Laminet & Pluckthun, 1989; Zahn et al., 1994b). The cysteines of PLared are very resistant to oxidation or alkylation, indicating that the native-like structure of PLared traps the cysteine thiols in a sterically inaccessible state. The X-ray crystal structure of native pLa (DeLucia et al., 1980) shows that the single disulfide bond is buried in the interior of the molecule. The native-like activity and the resistance to alkylation suggest that the cysteine residues of PLared are inaccessible as well. Our results suggest that PDI may provide multiple strategies for dealing with kinetic traps. Under second-order conditions, the PDI-dependent oxidation of /?Laredis approximately 500-fold faster than oxidation by GSSG. Most of this rate acceleration results from the high kinetic reactivity of PDI’s disulfides that allows relatively low concentrations of PDI to efficiently trap PLaredevery time it spontaneously unfolds. Because of its enhanced reactivity and its high local concentration in the ER (Lyles & Gilbert, 1990b; Zapun et al., 1992), PDI-facilitated disulfide formation may avoid kinetic traps by reacting with cysteines before they become trapped. If kinetic traps do form, PDI’s high kinetic reactivity as an oxidant also diminishes the significance of unreactive, sterically shielded cysteines as long as unfolding does not limit the rate. Covalent and noncovalent interactions with PDI are insufficient to promote a significant extent of PLaredunfolding. In addition, high concentrations of PDI or other chaperones do not increase the rate of PLared unfolding. If this is true in the cell, the inability to accelerate unfolding would imply that a sufficiently slow unfolding rate may help define a structure as “native”.
Biochemistry, Vol. 34, No. 41, 1995 13643
MATERIALS AND METHODS Materials. Recombinant rat PDI was expressed in Escherichia coli strain BL21(DE3) and purified to ’95% homogeneity as described previously (Gilbert et al., 1991). RTEM-1 P-lactamase was a generous gift of Dr. Timothy Palzkill (Baylor College of Medicine, Houston, TX). Glutathione (GSH), glutathione disulfide (GSSG), N-ethylmaleimide (NEM), iodoacetamide (IAM), dithiothreitol (DTT), bovine serum albumin (BSA), and lysozyme were from Sigma (St. Louis, MO); 2-mercaptoethanol was from J. T. Baker (Phillipsburg, NJ). Guanidine hydrochloride (Gdn.HC1) was from Pierce (Rockford, IL). The /3La substrate, nitrocefin, was obtained from Becton-Dickenson Microbiological Systems (Cockeysville, MD). GroEL/ES and recombinant hamster Grp78 (BiP) were obtained from Stress-Gen (Vancouver, BC). Methods. pLa (0.25 mg/mL) was reduced and denatured in 100 mM Tris, pH 8.0, 6.4 M Gdn.HC1, and 20 mM 2-mercaptoethanol for 1.5 h at 37 “C. The reduced and denatured enzyme had less than 5% the activity of the native enzyme. Reduced, refolded P-lactamase @Lared) was produced by diluting the reduced, denatured enzyme 50-fold into 0.1 M Tris, pH 8.0, 25 “C. After 15 min, the refolded, active enzyme was further diluted into alkylation or oxidation reactions. Residual thiol (
